A linkage map for sugi (Cryptomeria japonica) based on RFLP,RAPD, and isozyme loci

A linkage map for sugi was constructed on the basis of restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), and isozyme loci using a three-generation pedigree prepared for genetic analysis of heartwood color. A total of 128 RFLP (123 cDNA and 5 genomic probes), 33 RAPD, 2 isozyme, and 1 morphological (dwarf) loci segregated in 73 progeny. Of the 164 segregating loci, 145 loci were distributed in 20 linkage groups. Of these loci, 91 with confirmed map positions were assigned to 13 linkage groups, covering a total of 887.3 cM. A clustering of markers with distorted segregation was observed in 6 linkage groups. In the four clusters, distortions with a reduction in the number of homozygotes from one parent only were found.Abbreviations MAS marker-assisted selection - PAGE polyacrylamide gel electrophoresis - QTL quantitative traits of loci - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism This work was supported by a Grant-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan (Cooperative Research, no. 04304017)     

Assignment of RFLP linkage groups to their respective chromosomes in aneuploids of sugi (Cryptomeria japonica)

Aneuploids of sugi (Cryptomeria japonica) were found in the open-pollinated progenies of triploidplus tree clones. Seven trisomics and one hypotriploid were used to assign the chromosomes to the RFLP linkage groups constructed previously. The Southern blots containing their genomic DNA were hybridized with the labeled DNA clones corresponding to the loci in the linkage map. The additional dosage in autoradiographs showed that the cloned DNA fragment was located on the extra chromosome in the trisomics. On the other hand, the extra chromosome in two trisomics and the chromosome lacking the triplet in the hypotriploid were cytologically identified as chromosome 10 by consistent presence of a secondary constriction in the proximal region of its short arm. As a result, three linkage groups were assigned to their respective chromosomes, namely chromosome 10 and two other chromosomes.     

Physical map of chloroplast DNA of aerial yam,Dioscorea bulbifera L.

Summary A physical map of chloroplast DNA (ctDNA) of aerial yam, Dioscorea bulbifera L. was constructed using three restriction endonucleases, PstI, SalI, and SmaI. In addition, a clone bank of the BamHI-digested fragments were generated, and the locations of most BamHI fragments on the map were also determined. The ctDNA of D. bulbifera was found to be a circular molecule with a total size of ca. 152 kb involving two inverted repeats of ca. 25.5 kb, and small and large single copy regions of ca. 18.5 and 83.4 kb, respectively. The genes for the large subunit of the ribulose 1,5-bisphosphate carboxylase (rbcL) and the ATP-synthase subunits and (atpB/atpE) were mapped.Contribution from the Plant Germ-plasm Institute and the Laboratory of Genetics (No. 504), Faculty of Agriculture, Kyoto University, Japan. The work was supported in part by a Grant-in-Aid (No. 60400005) from the Ministry of Education, Science and Culture, Japan     

The physical map of the chloroplast DNA from Asparagus officinalis L.

The genus Asparagus consists of 100–300 species of both dioecious and hermaphrodite plants. Since there are diploid, tetraploid, and hexaploid plants in this genus, RFLP (restriction fragment length polymorphism) analysis of chloroplast DNA (ctDNA) is suitable for examining the phylogenetic relationships. We have constructed a physical map of the ctDNA of garden asparagus (A. officinalis L. cv Mary Washington 500 W) using five restriction endonucleases, namely, BamHI, PstI, SalI, HindIII, and XhoI. Asparagus ctDNA was digested with restriction enzymes and cloned into plasmid and phage vectors, and a clone bank was constructed that covered 70% of the genome. A physical map was constructed by Southern hybridization of total DNA from asparagus with homologous and heterologous probes. The asparagus ctDNA was about 155 kb long and it contained two inverted repeats (23kb each) separated by a large single-copy region (90kb) and a small single-copy region (19kb). Fifteen genes, encoding photosynthesis-related proteins, rDNAs, and tRNAs, were localized on the physical map of asparagus ctDNA. Comparing the length and the gene order of asparagus ctDNA with that of other plants, we found that asparagus ctDNA was similar to tobacco ctDNA but different from rice ctDNA. The restriction patterns of the ctDNAs from several varieties of A. officinalis and three species of Asparagus were analyzed. The restriction patterns of the varieties of A. officinalis were very similar, but polymorphisms were detected among the three species of Asparagus.     

Formation and vertical distribution of sapwood and heartwood in Cryptomeria japonica D. Don

The formation and vertical distribution of sapwood and heartwood were studied with a 45-year-old Cryptomeria japonica D. Don. The tree was grown at a plantation with 1.5 m × 3.0 m spacing near Miao-Li, Taiwan and was felled on 27 February 1992. The thickness of sapwood and heartwood was expressed by a ring count and a linear measurement. The east-west (E-W) wood strips were collected from 0.3 m above ground upwards to the top of the tree at 2.5 m intervals. The sapwood thicknesses from the base to the 10.3 m tree level height are around 20–22 growth rings and 42±2 mm. At the top of the tree, the sapwood thickness is narrower. The heartwood, which decreases in thickness with increasing tree level heights is not found at the top of the tree. The heartwood appears as a conical shape in the tree trunk. There is no statistical difference in sapwood/heartwood thickness between E-W aspects. Tree level heights and the tree level age were found to be important parameters in determining the thickness of sapwood/heartwood.     

Restriction endonuclease cleavage site map of safflower (Carthamus tinctorius L.) chloroplast DNA

Summary The restriction endonucleases SalI, PstI, KpnI and HindIII have been used to construct a physical map of safflower (Carthamus tinctorius L.) chloroplast DNA. This was accomplished by hybridizing Southern blots of single and double digested chloroplast DNA with ³²P-dCTP nick-translated SalI, KpnI and HindIII probes which were individually isolated from agarose gels. The chloroplast DNA was found to be circular and to contain approximately 151 kbp. In common with many other higher plant chloroplast DNAs a sequence of about 25 kbp is repeated in an inverted orientation. The small and large single copy regions separating the two repeated segments contain about 20 kbp and 81 kbp, respectively. The rRNA structural genes were also mapped by Southern blot hybridization and are co-linear with several other plant species.     

Physical maps of Nicotiana chloroplast DNA constructed by an efficient procedure

Summary The restriction profiles of chloroplast DNA (cpDNA) from Nicotiana tabacum, N. sylvestris, N. plumbaginifolia, and N. otophora were obtained with respect to AvaI, BamHI, BglI, HindIII, PstI, PvuII, SalI, and XhoI. An efficient mapping method for the construction of cpDNA physical maps in Nicotiana was established via a computer-aided analysis of the complete cpDNA sequence of N. tabacum for probe selection. The efficiency of this approach is demonstrated by the determination of cpDNA maps from N. sylvestris, N. plumbaginifolia, and N. otophora with respect to all of the above restriction endonucleases. The size and basic structure of the cpDNA from the three species are almost identical, with an addition of approximately 80 bp in N. plumbaginifolia. The restriction patterns and hence the physical maps between N. tabacum and N. sylvestris cpDNA are identical and there is no difference in the Pvull digests of cpDNA from all four species. Restriction site variations in cpDNA from different species probably result from point mutations, which create or eliminate a particular cutting site, and they were observed spanning the whole chloroplast molecule but highly concentrated in both ends of the large, single-copy region. The results presented here will be used for the forthcoming characterization of chloroplast genomes in the interspecies somatic hybrids of Nicotiana, and will be of great value in completing the exploration of the phylogenetic relationships within this already extensively studied genus.     

Immunocytochemical localization of the allergenic proteins in the pollen of Cryptomeria japonica

Applying an immunocytochemical method, a localization of the protein Cry j I in the Cryptomeria japonica pollen, which is the major allergen responsible for Japanese cedar pollinosis, is investigated with the monoclonal and polyclonal antibodies produced from the protein. The protein that reacts to the polyclonal antibody localizes on the sexine, nexine, between nexine and intine layers, orbicles, cell wall of a generative cell, Golgi body and Golgi vesicles. The allergenic protein contained in the exine and orbicles of Japanese cedar pollen can diffuse or dissolve easily from there into the mucus covering of the eye and nose, causing a response in less than 1 min after exposure. Since the orbicles have a diameter of about 430 nm, they can pass easily through the pores of most protective masks to reach the sensitive tissues of the patient. The proteins react to the monoclonal antibodies (J1BO1 and J1BO7) and localize on the Golgi body, sexine, nexine and orbicles (but not between the nexine and intine layers), and on the generative cell wall. In the young pollen grain, numerous allergenic protein particles contained in the orbicles and sexine layer, but there is only a small amount of the protein between the nexine and intine layers, since the intine layer is not yet complete at this stage. More will be accumulated there during developmental maturation. The allergenic protein is also found on the tapetal materials remaining in the young anther. Since the materials forming the exine layer and orbicles come from tapetal tissue, it is assumed that some of the allergenic protein is produced in the tapetum and localized in the orbicles and pollen wall during maturation, and that the rest of the allergenic protein is produced in the Golgi body in the mature pollen grain.     

A histological comparison of the development of pollen and female gametophytes in fertile and sterile Cryptomeria japonica

To determine a possible mechanism causing male and female sterility in Cryptomeria japonica male and female cones were collected from a C. japonica, tree, ShinDai2, that lacks pollen release and fertile seeds and specimens were processed to examine the development of pollen and female gametophytes using light microscopy and field emission scanning electron microscopy. Pre-meiotic development proceeded normally, but the formation of aberrant meiotic products was observed in cones of both sexes. In sterile microsporangia, heterogeneous microspore populations ranging from monads to polyads gave rise to mature pollen grains of non-uniform size. These pollen grains were covered with an amorphous layer and adhered to each other. In addition, they remained in the microsporangia and were not released even after the onset of pollen dissemination from fertile trees. In the ovules of sterile female cones, megaspores with abnormal shapes, numbers, and sizes formed, and the development of female gametophytes was arrested at the free nuclear or archegonium formation stages. These gametophytes collapsed, and no fertile embryo was generated. Results indicate that meiotic defects are important in the sterility mechanism.     

Clone bank and physical and genetic map of potato chloroplast DNA

Summary Clone banks of PvuII, BamHI and XhoI fragments were generated of the Solanum tuberosum cv Katahdin plastome. These clone banks, in conjunction with molecular hybridization to tobacco ctDNA probes, were used to construct a physical map of potato ctDNA. The potato plastome was found to be a circular molecule of 155–156 Kbp containing two inverted repeat regions of 23–27 Kbp. The arrangement of restriction sites is very similar to that of other Solanaceae plastomes. Heterologous hybridization to known ctDNA encoded gene probes from tobacco allowed us to establish a genetic map of the potato chloroplast genome. The arrangement of these genes on the potato plastome resembles that on most higher plant ctDNAs.     

Nicotiana chloroplast genome III. Chloroplast DNA evolution

Summary Nicotiana chloroplast genomes exhibit a high degree of diversity and a general similarity as revealed by restriction enzyme analysis. This property can be measured accurately by restriction enzymes which generate over 20 fragments. However, the restriction enzymes which generate a small number (about 10) of fragments are extremely useful not only in constructing the restriction maps but also in establishing the sequence of ct-DNA evolution. By using a single enzyme, Sma I, a elimination and sequential gain of its recognition sites during the course of ct-DNA evolution is clearly demonstrated. Thus, a sequence of ct-DNA evolution for many Nicotiana species is formulated. The observed changes are all clustered in one region to form a hot spot in the circular molecule of ct-DNA. The mechanisms involved for such alterations are mostly point mutations but inversion and deficiency are also indicated. Since there is a close correlation between the ct-DNA evolution and speciation in Nicotiana a high degree of cooperation and coordination betwen organellar and nuclear genomes is evident.     

Cleaved amplified polymorphic sequence markers in sugi, Cryptomeria japonica D. Don, and their locations on a linkage map

Sugi, Cryptomeria japonica D. Don, is one of the most important forestry species in Japan. We here report the development of 217 CAPS markers derived from sugi cDNA libraries. More than half of a set of STS markers produced could be converted into CAPS markers using restriction endonuclease analysis. Of the 217 markers, 71 showed different patterns of polymorphism when they were digested with a range of endonucleases and, in total, 347 polymorphisms were found in the various combinations of STSs and endonucleases. When the polymorphisms gave co-dominant patterns in a screening program, the polymorphic information content (PIC) used to evaluate the value of the polymorphisms was relatively high (0.33, on average) compared to the information yielded by commonly used markers, like isozymes. The results of a segregation analysis suggest that approximately 80% of the CAPS markers developed here will show co-dominant inheritance. From logistic regression analysis, the polymorphisms were found to be associated more strongly with intron than with exon regions. Sixty two markers were subsequently mapped on the previously reported linkage map, 15 of which showed abnormal segregation, presumably caused by linkage with lethal factors. Received: 7 December 2000 / Accepted: 23 January 2001     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Summary Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system     

Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenic tissue

A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on hygromycin and kanamycin medium, respectively. Transgenic plants were regenerated through somatic embryogenesis in 4 lines out of these 11 lines. Green fluorescent protein fluorescence was observed under fluorescent microscopy. Integration of the genes into the genome was confirmed by polymerase chain reaction analysis of embryogenic tissues and Southern blot analysis of regenerated plantlets.     

Live crown tops of Japanese cedar (Cryptomeria japonica) approach one another in height at urban forest sites

Since the 1980s, we have found Japanese cedar, Cryptomeria japonica (L. f.) D. Don, trees with crown tops affected by dieback in isolated urban forests on the low altitude plain. To clarify future growth of C. japonica in these forests, we investigated their growth, decline levels and water status. The live crown-top heights from the ground (LCTHs) per diameter at breast height (dbh) were lower in forests with C. japonica top dieback than in forests with no top dieback. Furthermore, in a forest with C. japonica top dieback, the LCTHs were similar between trees although dbh and decline levels were different. Moreover, water status near the top of the live foliage was very similar although decline levels were different, suggesting that in urban forests, where C. japonica top dieback is observed, the LCTH is subject of restriction. A sudden increase in temperatures since the 1980s may be restricting the LCTHs of C. japonica in urban forests. We conclude that LCTHs of C. japonica in urban forests are becoming lower and more equal in each forest. If temperature continues to increase, height restriction will become more severe and LCTHs of C. japonica in urban forests will become even lower.     

Selection and microculture of single embryogenic cell clusters in japanese conifers: Picea jezoensis, Larix leptolepis and Cryptomeria japonica

Summary A microculture method for single embryogenic cell clusters was established for several Japanese conifer species, namely Picea jezoensis, Larix leptolepis and Cryptomeria japonica. Individual small, dense cell clusters were identified and picked up by a micromanipulator and cultured in 50 μl liquid medium in a 96-well culture plate. In all three species studied, a majority of the cell clusters actively proliferated within the wells. Maturation of somatic embryos was successful when the newly proliferated cell clusters were transferred to solid abscisic acid-containing medium. Thus, the small, dense cell clusters could be a useful morphological marker for cells capable of proliferation and differentiation.     

A diterpene quinone from the bark of Cryptomeria japonica

A diterpene, cryptoquinone, was isolated from the bark of Cryptomeria japonica, the structure, 7,11,14-trioxoabieta-8,12-diene, was established by spectral analyses and X-ray crystallography. This diterpene quinone showed moderate antifungal activities against Pyricularia orizae and Alternaria alternata, and cytotoxic activity against mouse lymphoid neoplasm (P388) cells with an IC(50) of 0.26 microg/ml.     

Immunohistochemical localization of agatharesinol,a heartwood norlignan,in Cryptomeria japonica

Localization of a heartwood norlignan, agatharesinol, in Sugi (Japanese cedar, Cryptomeria japonica D. Don, Taxodiaceae) was investigated by immunohistochemistry. Immuno light microscopy showed that the contents of ray parenchyma cells were immunostained in heartwood but not in sapwood. The staining of the heartwood tissue was competitively inhibited by agatharesinol but not by other Sugi heartwood extractives, and was, furthermore, markedly reduced by pre-extraction of the tissue with MeOH. These results indicated that the staining can be ascribed to the immunolabeling of agatharesinol in situ. The accumulations over the inner surface of some tracheid cell walls adjacent to the ray parenchyma cells were also immunolabeled, while the contents in axial parenchyma cells were not. In conclusion, agatharesinol was localized in the ray parenchyma cells in Sugi heartwood, and differences between the chemical structure of the contents of ray and axial parenchyma cells were also suggested.